Project Summary Macrophages are a diverse family of professional phagocytes and `accessory cells' present within most tissues, and equipped with long and motile filopodia that continuously explore surrounding matrix and cells. Macrophages recognize and scavenge pathogens as well as unfit cells and cell debris, glycoproteins and lipids, and produce a large range of bioactive molecules and growth factors. A literature that covers 150 years of research has demonstrated their important and complex roles (i) as innate immune sentinels for the uptake of pathogens, but also as (ii) regulators of epithelia and blood and lymphatic vessels morphogenesis during fetal development, postnatal homeostasis and tissue remodeling, and inflammatory and degenerative processes. An overarching hypothesis that drives the present proposal and the work of our laboratory is that the understanding the genetic and molecular events that control development, growth, and maintenance of macrophages within tissues is essential to elucidate their roles in disease and the consequence(s) of genetic polymorphisms. Early studies suggested that tissue macrophages originate and renew from hematopoietic stem cells (HSC) via circulating progenitors such as blood monocytes. However, as recently shown by us and others, monocytes and macrophages play different roles in disease pathogenesis, and in fact, they represent distinct cell types, as most tissue-resident macrophages develop from embryonic progenitors distinct from HSCs and are maintained in postnatal tissues independently of bone marrow progenitors and monocytes. Furthermore, macrophages that reside in distinct tissues present with different molecular identities and functions. The tissue-specific identities of macrophages are in part maintained by their local microenvironment, but are also likely to result from developmental processes. The present project builds on these exciting and `paradigm-changing' developments. 1) We will characterize the genetic and molecular determinants that control macrophage development from embryonic progenitors in the mouse, using sate-of-the-art fate mapping methods, and explore the mechanisms that underlie the colonization of embryos by the macrophages precursors. 2) We will also take advantage of single-cell genomic and systems biology approaches to characterize the mechanisms and spatio-temporal sequence of events responsible for tissue macrophage specification in vivo. We will further test the role of novel key transcriptional regulators that control macrophage differentiation, maintenance, and functions in postnatal tissues, with a focus on Kupffer cells. Along this process we will generate novel genetic tools needed to distinguish and selectively target tissue-resident macrophages, independently of HSC and their progeny. Altogether, this work represents a ground-breaking advance toward our aim to characterize the function of tissue-resident macrophage in health and disease.